Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors ...

Cancer Therapy: Preclinical

Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589 David Z. Qian,1 Yukihiko Kato,1 Shabana Shabbeer,1 Yongfeng Wei,1 Hendrik M.W. Verheul,1 Brenda Salumbides,1 Tolib Sanni,1 Peter Atadja,2 and Roberto Pili1

Abstract

Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589. Materials and Methods:To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo.To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we didWestern blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo. Results: LBH589 induced acetylation of histone H3 and a-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G2-M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth. Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

Histone deacetylase (HDAC) inhibitors represent an emergent

including short-chain fatty acids (i.e., sodium butyrate), hydroxamic acids (i.e., thrichostatin A, SAHA, and LAQ824), cyclic peptides containing a 2-amino-8-oxo-9,10-epoxy-decanoyl moiety (i.e., trapoxin A), cyclic peptides without the 2-amino8-oxo-9,10-epoxy-decanoyl moiety (i.e., FK228), and benzamides (i.e., MS-275). These agents induce a dose-dependent inhibition of either class I (1, 2, 3, and 8) or class II (4-10) HDACs, or both. Depending on the specific drug, the cancer cell line tested, the dose, and time of exposure, HDAC inhibitors can induce G1 or G2 cell cycle arrest and/or cell death (3, 4). The reported p53-independent induction of the p21 gene and protein expression is probably due to the nature of epigenetic regulation of the p21 promoter and, along with histone lysine acetylation, represents a common molecular observation associated with drug exposure (5, 6). There have also been reports of up-regulation of proapoptotic pathways and downregulation of prosurvival pathways associated with the HDAC inhibitors, which depend on the agent and the tumor cell line used (7, 8). We have previously shown that the HDAC inhibitors phenylbutyrate and MS-275 induce the expression of the putative tumor suppressor gene RARb2 and increase the retinoid sensitivity in retinoid-resistant prostate and renal cell carcinoma cell lines (9, 10).

class of anticancer drugs. The targets of these agents are enzymes (HDACs), which induce histone and nonhistone protein deacetylation at the lysine residues, chromatin condensation, and modulation of gene expression. Altered HDAC activity is associated with cancer, and inhibitors targeting HDACs have been shown to induce tumor cell cytostasis, differentiation, and apoptosis in various hematologic and epithelial tumors (1, 2). Five classes of HDAC inhibitors have been characterized,

Authors’ Affiliations: 1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland and 2 Novartis Institute for Biomedical Research, East Hanover, New Jersey Received 5/23/05; revised 10/14/05; accepted 10/28/05. Grant support: Prostate Cancer Foundation Award, Sidney Kimmel Research Award (R. Pili), and Aegon fellowship (D. Qian). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Roberto Pili, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1M52, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-502-7482; Fax: 410-614-8160; E-mail: piliro@ jhmi.edu. F 2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1132

Clin Cancer Res 2006;12(2) January 15, 2006

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HDAC Inhibitors and Tumor Angiogenesis

Cell viability assay. The effect of LBH589 on HUVEC growth and survival was assessed by a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide inner salt assay (Roche, Indianapolis, IN) as previously described (6). In brief, 1
104 cells were seeded into 96-well plates. After an overnight incubation, the medium was replaced by fresh EGM-2 with either complete growth factors (EGM-2 bullet kit), 50 ng/mL VEGF-A (R&D Systems, Minneapolis, MN), or 50 ng/mL basic fibroblast growth factor (bFGF; R&D Systems). LBH589 was added at 0 to 800 nmol/L final concentrations. After 48 hours of incubation, the viable cells (a net result of both cell growth and cell death) were measured by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide inner salt reagent at UV 490 nm as described by the manufacturer’s protocol. The UV readings of solventtreated controls were normalized to 100%, and the readings from LBH589-treated cells were expressed as % controls. Cell proliferation assay. The proliferation assay was run as previously described (3, 6). Briefly, cancer and endothelial cells were seeded (1
104 per well) in six-well plates and incubated at 37jC, 5% CO2 for 24 to 48 hours. The medium was then replaced with appropriate basal medium without growth factors. After overnight starvation, cells were counted in triplicate with a Coulter counter and designated as day 0 values. Then, cells were treated with different concentrations of LBH589 (triplicate) in complete media with growth factors and serum. Forty-eight hours later, the viable cells from each condition were harvested and counted by Coulter counter. Cell number at day 0 was normalized to 100%, and cell proliferation following 48 hours of treatment was adjusted to the % day 0 controls. The experiments were repeated thrice with similar results. Cell cycle and cell death analysis. Proliferating HUVECs were starved overnight and then treated with increasing concentrations of LBH589 with complete media for 24 hours. After drug treatment, both starved and treated cells were harvested, fixed, and stored in 70% ice-cold ethanol. The percentage of the cell cycle that was in G0G1, S, and G2-M phase was quantitated by using a propidium iodide – based cellular DNA flow cytometry analysis kit (Roche) following manufacturer’s instructions. For cell death analysis, Annexin V and propidium iodide costaining followed by flow cytometry was done according to manufacturer’s instructions (BD Biosciences, San Jose, CA). Matrigel angiogenesis assay in vitro (tube formation). HUVECs were cultured in complete EGM-2 media before being plated in 24-well plates (5
104 per well) previously coated with 300 AL of growth factor – reduced Matrigel (BD Biosciences), in the presence of LBH589 or solvent control. The morphology of capillary-like structures formed by HUVECs 15 hours after culturing was visualized using an inverted microscope (Zeiss Axioskop) and photographed with a digital camera. Tube formation was analyzed with an imaging system (Image-Pro) as previously described (6). Matrigel invasion assay. The Matrigel invasion assay was done with precoated Matrigel inserts following manufacturer’s instructions (BD Biosciences). Proliferating HUVECs were pretreated with indicated doses of LBH589 overnight, and then the cells were harvested and resuspended in basal EGM-2 media with 0.5% fetal bovine serum and the indicated dose of LBH589. An equal number of cells (4
105) were added to the invasion chambers in triplicate. The chemoattractants were either VEGF-A or SDF-1a (50 ng/mL, R&D Systems). Twenty-four hours later, the membranes containing migrated cells were fixed and stained, and cell numbers were counted under a microscope. Five random fields were chosen for each membrane, and the results were expressed as migrated cells per field for each condition. Reverse transcription-PCR and Western blot analysis. The details of reverse transcription-PCR and Western blots have been described previously (6). The antibodies for AKT and extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphorylation were purchased from Cell Signaling (Beverly, MA); antibodies for HIF-1a and CXCR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies for histone H3 and acetylated H3 were purchased from

In addition to the inhibitory effect on cancer cell proliferation, HDAC inhibitors have been reported to inhibit the process of new capillary blood vessel formation or angiogenesis (6, 11 – 13). Tumor-initiated angiogenesis takes place following an ‘‘angiogenic switch.’’ During the initial avascular tumor growth, there is an accumulation of hypoxia-inducible factor1a (HIF-1a) protein, which activates an array of gene transcriptions, including vascular endothelial growth factor (VEGF), one of the most proangiogenic factors (14). VEGF-A (the principal isoform of VEGF) binds to VEGF receptor 2 (VEGFR2) on the surface of quiescent endothelial cells in the surrounding vessel walls and triggers the signaling pathways for endothelial activation and downstream angiogenesis (15). During the multistep angiogenesis process, several ligand/ receptor interactions are required. The interaction of angiopoietins and endothelial receptor Tie-2 affects endothelial cell survival and vessel stability (16). Stromal-derived factor-1 (SDF-1) and endothelial chemokine receptor CXCR4 influence the homing of mobilized/activated endothelial cells or endothelial progenitor cells to active sites of neovascularization (17, 18). Overall, the combined net result of ligand/receptor interaction activates intracellular pathways, leading to endothelial cell survival, proliferation, mobilization, and invasion of the extracellular matrix. This culminates in recognition and homing to the sites of new blood vessel formation. In our previous work, we identified two different HDAC inhibitors, phenyl butyrate (short-chain fatty acid) and LAQ824 (hydroxamic acid), which have antiangiogenesis activity both in vitro and in vivo (3, 6). Angiogenesis inhibition induced by LAQ824 was associated with modulation of angiogenesisrelated genes both in cancer cells (inhibition of HIF-1a and VEGF) and in endothelial cells (inhibition of Tie-2 and survivin, an inhibitor of apoptosis). Other groups have also reported that other HDAC inhibitors, including SAHA, TSA, FK228, valproic acid, and apicidin, have antiangiogenic activity (11 – 13). In the current study, we investigated the antiangiogenic effect of a recently developed HDAC inhibitor, LBH589 that is currently in phase I clinical trial (19, 20). We identified altered gene expression and protein phosphorylation induced by LBH589 in endothelial cells. This data lead us to explore the potential of LBH589 as therapeutic agent with dual activity against both tumor proliferation and angiogenesis.

Materials and Methods Cell lines, reagents, and animals. Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Walkersville, MD) and cultured in complete EGM-2 medium containing EGM-2 bullet kit (Cambrex) with growth factors and 2.5% fetal bovine serum supplied by the vendor. All experiments were done using endothelial cells between passages 3 and 8. A human prostate (PC-3) carcinoma cell line was originally obtained from the American Type Culture Collection (Manassas, VA), maintained in RPMI 1640 with l-glutamine supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). LBH589 was kindly provided by Novartis Pharma AG (Cambridge, MA). Stock solutions for in vitro assays were prepared in DMSO with final concentrations of 400 nmol/L (Fig. 2D). Costaining of cells with Annexin V and propidium iodine followed by flow cytometry analysis confirmed a significant cell death in HUVECs but not in PC-3 following treatment with LBH589 (Fig. 2E). LBH589 inhibited in vitro angiogenesis. In the presence of high concentrations of VEGF-A, endothelial cells form tube and capillary-like structures on the surface of basement membrane matrices (Matrigel), through a process involving attachment, alignment, and migration. Treatment with a noncytotoxic dose of LBH589 (50-200 nmol/L) significantly reduced this process (Fig. 3A). We also tested the effect of LBH589 on endothelial chemotaxis. Proliferating HUVECs were starved for 24 hours and pretreated with different concentrations of LBH589 for the last 12 hours of starvation. The cells were then tested in the

growth was evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H -tetrazolium-5-carboxanilide inner salt assay (Fig. 2A). These results showed that LBH589 had similar dose-dependent inhibitory effects on HUVECs regardless of the growth factor used to induce cell proliferation. Using the complete EGM-2 bullet kit growth media, the inhibition of endothelial cell proliferation was also determined by counting viable cell numbers as previously described (6). Following 48 hours of treatment, the solvent-treated control HUVECs had a 1.5-fold increase in cell number, whereas V200 nmol/L LBH589 inhibited cell growth. Cytotoxicity was observed with z400 nmol/L LBH589 (Fig. 2B) as evidenced by a sharp decrease in cell number. This dose-dependent response was rather unique for endothelial cells. LBH589 inhibited cell proliferation in LNCaP and PC-3 human prostate carcinoma cells at concentrations up to 1 Amol/L (Fig. 2C) without significant cell death and induced primarily G2-M cell cycle arrest (data not shown).

Fig. 2. Effect of LBH589 on HUVEC proliferation and viability. A , proliferating HUVECs, cultured in complete media (EGM-2 bullet kit), or 50 ng/mL VEGF-A, or 50 ng/mL of bFGF, were treated with increasing concentrations of LBH589 for 48 hours. Cell growth and viability were measured by 2,3-bis[2-methoxy-4-nitro5-sulfophenyl]-2H-tetrazolium-5carboxanilide inner salt assay. Points, mean % solvent-treated control (n = 6); bars, SD. B-C, proliferating HUVECs and prostate carcinoma cells PC-3 were counted by Coulter counter and treated with the indicated doses of LBH589 for 48 hours. The viable cells were counted and expressed as % day 0 control. The cell numbers at the beginning of incubation (day 0) were normalized to 100%. For treated and untreated cells after 48 hours, cell number >100% meant net cell gain,